System and method for solution based multiparameter analysis of analytes

ABSTRACT

There is described a system for multiparameter analysis of analytes. The system comprises: 1) primary supports ( 1 ) with a largest dimension ( 3 ) of 500 μm or less suspended in use in a fluid solution; 2) each primary support ( 1 ) comprises an identification means ( 2 ) for identification thereof; 3) at least one primary analyte ( 12 ) is bound to each primary support ( 1 ); 4) a secondary analyte ( 12 ′) is suspended in use in the fluid solution; and 5) a measuring means ( 25 ) is arranged in communication with the fluid solution for monitoring interaction between the primary analyte ( 12 ) and secondary analyte ( 12 ′). The system is distinguished in that: 6) secondary supports ( 1 ′) with a largest dimension at the most the same size as the largest dimension ( 3 ) of the primary supports ( 1 ) are suspended in use in the fluid solution; 7) each secondary support ( 1 ′) comprises an identification means ( 2 ′) for identification thereof; 8) at least one secondary analyte ( 12 ′) is bound to each of the secondary supports ( 1 ′); and 9) the measuring means ( 25 ) is arranged to detect any post-reaction interaction between one or more primary analyte ( 12 ) and one or more secondary analyte ( 12 ′) by detecting the identification means ( 2, 2 ′) of the primary and secondary supports ( 1, 1 ′) attached thereto. There is also described a method of multiparameter analysis of analytes using the system.

FIELD OF THE INVENTION

The present invention relates to systems for multiparameter analysis ofanalytes in solution; moreover, the invention also concerns a method ofperforming such multiparameter analysis of analytes in solution.

BACKGROUND TO THE INVENTION

There are many industries in which there is a requirement to studyhundreds or thousands of samples simultaneously. Using traditionalmanual techniques of serial testing, such study has proved to be verytime-consuming and expensive. Multi-parameter screening has hence becomean important tool for processes in which rapid testing of many samplesis required. For example, recent advances in our understanding of thehuman genome have led to a huge increase in the number of many noveldrug targets being identified. At the same time, breakthroughs in theautomated synthesis of chemical compounds has led to the availability ofsubstantial libraries of compounds to be screened for possiblepharmaceutical activity at these novel targets. Taken together, suchdevelopments have created a growing demand for more rapid, inexpensiveand less labour-intensive analysis methods for drug discovery anddevelopment. Further examples of industries where multiple testing hasfound applications are in diagnostics, proteomics, the food industry(e.g. for detecting veterinary drug residues in foods, for monitoringundesirable and dangerous pathogens, and for identity preservation), andthe cosmetics industry (e.g. for providing alternatives to animaltesting, for screening active ingredients and novel molecules).

The development of multiplexing technologies has improvedhigh-throughout screening processes. There are two main strategiesemployed, namely either physically separating each molecule to aparticular place on a microarray (e.g. labelled tubes or wells, highdensity arrays or microchips), or performing reactions on individuallyencoded microcarriers, each carrier having a particular molecule boundto its surface.

In the first strategy, it is the exact location (x,y-coordinate) on themicroarray that allows for identification of a target/compound which isanalysed at that place. This method of tracking a reaction is usuallyreferred to as positional or spatial encoding. Many different microarrayformats are available commercially, e.g. the GeneChip® from Affymetrix.Characteristics of molecules being analysed on such microarrays mustoften be known and isolated beforehand; such prior knowledge makes it acomplicated and costly process to manufacture specific microarrays tocustomer requirements with short lead times. A further disadvantage ofspotted microarrays is the poor quality of the spotted molecule on themicroarray. This results in low reliability of test results therebyobtained from the arrays. Such low reliability has, in turn, resulted inextensive quality control requirements during manufacture of themicroarrays and spot arrays, or even high redundancy of each moleculebuilt into the microarray, to ensure the quality of spotting. With thenumber of tests on each microarray increasing, use of advancedminiaturisation is required. Miniaturisation has also been used todecrease the amount of reagents. In addition, many companies andresearch institutes use homebrew methods for producing microarrays. Thenumbers of tests that can be performed on these home brews are verylimited and also have the drawbacks described above. The reading ofthese homebrews is time and labour intensive with respect to the numberof data points read.

In the second strategy, namely the second method of microcarrier-basedassays, there arises a need to label each of the microcarriers (alsocalled supports) to allow for identification of the molecules bound totheir surface. This method allows for greater customisation by mixingthe uniquely encoded microcarriers in one reaction vessel and subjectingthem to an assay simultaneously. Those microcarriers that show afavourable reaction between the attached molecule and the target analytemay then have their code read, thereby leading to the identity of themolecule that produced the favourable reaction. An example of such atechnology is Luminex Corporation's xMAP™ technology. The xMap systemhas a limitation of 100 differently optically coded microcarriers.

When the number of tested molecules on individual microcarriersincreases advanced fluid handling is required. If the sameidentification code of a microcarrier is used for different molecules indifferent experiments there are contamination risks during thepreparation of the microcarrier with attached molecule. When the numberof microcarriers in an experiment increases, the problem of spectraloverlap adds further problems of false positives and poor data quality.Other examples of microcarrier technology employ multicolour encodingsimilar to the xMap system are, for example, Illumina's BeadArray™system, and Quantum Dot Corporation's Q-dot™ nanocrystals. Alternativemethods of identification coding microcarriers used in traditionalassays are the use of radio frequency identification (RFID) technology,e.g. PharmaSeq's micro-transponders described in a granted U.S. Pat. No.6,361,950, or barcode identification technology, e.g. SmartBeadTechnologies' UltraPlex™ system described in an international PCT patentapplication having a publication no. WO0016893. These new approaches ofencoding microcarriers have improved the signal-to-noise ratio ofdetecting such microcarriers.

Another encoded microcarrier solution includes the use of programmablematrices with memories as described in IRORI's published internationalPCT application no. WO 96/36436. This recording device stores theinformation of what molecules and biological materials are linked to thematrix material of each programmable matrice. These matrices can be insolution in one vessel or each linked to a well of a microtitreplate.Several matrices can also be arranged in an array taking the form of amicroarray. Other particle array solutions from Virtual Arrays Inc andUniversity of Hertfordshire are described in the published internationalPCT applications no. WO 00/63419 and WO 02/37944 respectively. Theseparticle based arrays allow greater customisation of the probes(molecules), which probes are attached to coded particle arrays andtested against a test sample, than the positional based microarraysolutions. These particle array solutions do require much automation androbotics when the number of multiplexed probes on uniquely identifiedparticle arrays becomes very large into the hundreds or even thousandsrange. All the particle based array solutions do also have problems withcross reactivity for certain analytes/molecules when the number ofmultiplexing increases. Reaction between the particle array labelledmolecules and a target analyte is detected using established detectionmethods like fluorescence, luminescence or radioactive labels, whichoften have limited shelf life.

Another system used to detect a target agent in a biological sample,described in an international PCT patent having a publication no.WO0242498, comprises a bead assay system that is read on opticalbiodiscs. This technology includes magnetic capture beads and reporterbeads. Both sets of beads are coated with probes, which arecomplementary to the target molecule sought in the biological sample. Ifthe target agent is present in the sample, the reporter bead and themagnetic capture bead binds to it. The bead complex is then isolatedusing a magnetic field and loaded onto an optical disc which has acapture layer affixed thereto. The presence of the dual bead complex canbe detected either electromagnetically or based on fluorescence. Thecombination of different sizes of magnetic beads and different types offluorescent reporter beads allows different target agents to be detectedsimultaneously. This dual bead systems experience many of the drawbacksdescribed for previous described microarray and microcarrier-based assaymethods of analysing target analytes with labour intense methodology,spectral overlap etc. They also require advanced sorting and readingequipment increasing the cost of their systems.

SUMMARY OF THE INVENTION

A first object of the invention is to provide an improved system for theanalysis of multiparameter analytes.

A second object of the invention is to provide a system to test largenumbers of multiple parameters simultaneously.

A third object of the invention is to improve the parallel testingthroughput of currently used microcarrier-based assay systems.

According to a first aspect of the invention, there is provided a systemas defined in the accompanying claim 1.

The system is of advantage in that it is capable of addressing at leastone of the aforementioned objects of the invention.

The system is beneficial in that it is flexible and can also be used tocomplement and/or improve existing support-based and/or microarraytechnologies. As all the reactions in the system are tagged by theinteraction of individual identifiable supports, the throughputpreviously achieved using support-based technology for tagging primaryanalytes with supports and testing against a fluorescent labelledsecondary analyte (e.g. the target analyte) is efficiently improved. Thepossibility of being able to test many primary analytes against manysecondary analytes drastically decreases the number of experiments, theamounts of reagents used and the increases the amount of multiparameterspossible to analyse simultaneously. Such improvement also allows the useof adapted conventional reading means, but requires the detection of theinteracting primary and secondary supports' identification means. Thepossibility of this multiparameter testing with interacting individuallyidentifiable supports substantially improves the analysis of for examplethe interactions of proteins or other large number of molecules with anumber of compounds. By using two different sets of identifiablesupports the benefit of these individually labelled supports become evenmore apparent. This allows the analysis of binding characteristics ofprimary and seconday analytes, previously difficult to achieve. Thesystem is hence not limited to being able to test high numbers ofprimary analytes against only a single or very few fluorescentlylabelled secondary analytes. This further allows System biologyexperiments previously only possible to be performed in silica to now beperformed empirically.

In a preferred embodiment of the invention, the primary supports are inthe form of microparticles decreasing the amount of reagents used foreach simultaneous testing process.

In a further preferred embodiment of the invention, the secondarysupports are the same size or smaller than the primary supports as thisallows improved possibility of quantification measurements of thesecondary analytes present in a sample.

In another preferred embodiment of the invention, the identificationmeans comprises one or more distinguishing geometrical features, such asshape, size, barcode or dotcode, enabling identification of eachsupport. This allows the use of well established identificationstandards such as for example barcodes which give good signal to noiseratio and decrease the risk of spectral overlap and false positives.

Other preferred embodiments of the invention, comprises the use of radiofrequency identification transponders (RFID) or optical identification,such as fluorescence or colour coding. The RFID gives the advantage ofvery large numbers of codes can be used and does not require visualcommunication between the measuring means and the identifiable support.The use of optical coding on the supports allows for combinations ofwavelengths or colours not possible with standard fluorescent markers,e.g. FITC labelled, and allows for using low cost labelled supports.

In a further embodiment of the invention, there is provided a solidsubstrate, which accommodates the liquid solution. This allows the useof the multiparameter analysis using interacting primary and secondarysupports to be used together with existing microarray technology for theanalysis of a three way interaction between analytes.

According to a second aspect of the invention, there is provided amethod as defined in the accompanying claim 15.

The method is of advantage in that it is capable of addressing at leastone of the aforementioned objects of the invention.

It will be appreciated that features of the invention can be combined inany combination without departing from the scope of the invention.

DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of exampleonly, with reference to the accompanying drawings wherein:

FIG. 1 is a plan view and a side view of a single support (microcarrier)comprising a sequential identification;

FIG. 2 is a schematic sectional side view of a single support(microcarrier) with analytes attached thereto;

FIG. 3 is a schematic diagram of a system for multiparameter analysis ofanalytes;

FIG. 4 a, b are schematic diagrams of the interaction between a primaryand a secondary support according to a preferred embodiment of themultiparameter analysis system;

FIG. 5 a, b are schematic diagrams of the interaction between a primaryand a secondary support according to an alternative embodiment of themultiparameter analysis system;

FIG. 6 is a schematic diagram of a system for multiparameter analysis ofanalytes comprising multiple supports and a fixed array or substrate;

FIG. 7 a, b are schematic diagrams showing the experiment reactionbetween supports, and a fixed array or substrate;

FIG. 8 is a schematic diagram illustrating a planar-based reader forinterrogating the system of FIG. 3; and

FIGS. 9 a, 9 b are schematic top views of a planar substrateillustrating examples of the measuring path taken by the planar-basedreader of FIG. 8.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

In FIG. 1, there is shown an illustration of a preferred embodiment of asupport for use in a system according to the invention. This preferredembodiment of the support is susceptible to being used as a primarysupport 1 and/or a secondary support 1′ in the system for multiparameteranalysis of analytes described further in the following detaileddescription. There is shown a single support 1, 1′; such a support willalso be referred to as a microcarrier, microparticle or “bead” in thefollowing description. The primary supports 1, 1′ can be fabricated fromvirtually any insoluble or solid material, for example one or more ofpolymers, silicates, glasses, fibres, metals or metal alloys. In thepreferred embodiment of the invention, the supports 1, 1′ are fabricatedfrom a metal, such as gold, silver, copper, nickel, zinc or mostpreferably aluminium. It is also preferable to use one or more polymers,such as polystyrenes, polyacrylates, polyamides, or polycarbonates whenfabricating the supports 1, 1′. The support 1, 1′ is preferably eitherpartially or totally coated in one or more of either of theabove-mentioned materials.

The support 1, 1′ incorporates an identification feature 2, 2′ which isalso referred to as an identification code or tag in the followingdescription. Examples of the identification features 2, 2′ may be basedon one or more of sequential identification, varied shape and size ofthe support, transponders (for example Radio Frequency IdentificationChips, RFIs) attached to the support, and fluorescent coding ordifferent colours of the support. Preferably, the identification feature2, 2′ is a sequential identification which can be in the shape of atleast one (or any combination thereof) of grooves, notches, depressions,protrusions, projections, and most preferably holes. The identificationfeature 2, 2′ being part of the support 1, 1′ is advantageous in thatthere is no need to label each support 1, 1′ after manufacture. Thesequential identification 2, 2′ is suitably a transmission opticalbarcode, which is machine readable, allowing enhanced signal to noiseratio if read in transmission or even reflection. An associatedsequential identification code is thereby recorded in the support 1, 1′as a series of holes using coding schemes similar to those found onconventional bar code systems, for example as employed for labellingmerchandise in commercial retailing outlets. Such a code allows the useof existing reader technology to determine the identification feature 2,2′ of the supports 1, 1′, thereby decreasing the initial investment whenadopting technology according to the invention.

In the preferred embodiment, the primary support 1 and/or secondarysupport 1′ is of substantially planar form with at least a principalsurface 6 as illustrated in FIG. 1. The support 1, 1′ has suitably awidth 4 to length 3 ratio in a range of circa 1:2 to circa 1:20,although a ratio range of circa 1:15 to circa 1:5 is especiallypreferred Moreover, the support 1, 1′ has a thickness 5 which ispreferably less than circa 3 μm, and more preferably less than circa 1μm. The thickness of less than circa 1 μm has been shown to providesufficient mechanical support strength for rendering the support 1, 1′useable in harsh experimental conditions. A preferred embodiment of theinvention concerns the support 1, 1′ having a length 3 of circa 100 μm,a width 4 of circa 10 μm and a thickness 5 of circa 1 μm; such a supportis capable of storing more than 100,000 different identificationsequence bar codes 2. Experimental demonstrations of up to 100,000different variants of the support 1, 1′ for use in bioassays for analytecharacterization experiments have been undertaken. The current barcoding systems used have error and directional checking and up to 32bits of information available on a support with a length of 100 μm. Thesupport 1, 1′ is susceptible to being fabricated in various lengths 3 ina range of 40 μm to 100 μm, and carrying between two and five decimaldigits of data in the sequential identification 2; examples of such asupport 1, 1′ have been fabricated for use in different experiments forthe detection of analyte characteristics.

Around 2.5 million supports similar to the support 1, namely primaryand/or secondary supports 1, 1′, may be fabricated on a single 3-inchdiameter semiconductor-type wafer, for example a silicon wafer, usingcontemporary established manufacturing techniques. Advantageously, theshape of the support 1, 1′ is such that it optimises the number ofsupports 1, 1′ manufactured per wafer and also substantially optimisesthe number of identification codes possible on the supports 1, 1′.Conventional photolithography and dry etching processes are examples ofsuch manufacturing techniques used to manufacture and pattern a materiallayer to yield separate solid supports 1, 1′ with bar-codedidentification 2, 2′.

A fabrication process for manufacturing a plurality of supports similarto the support 1, 1′ involves the following steps:

-   -   (1) depositing a soluble release layer onto a planar wafer;    -   (2) depositing a layer of support material onto the release        layer remote from the wafer;    -   (3) defining support features, including the sequential        identification 2, in the support material layer by way of        photolithographic processes and etching processes;    -   (4) removing the release layer using an appropriate solvent to        yield the supports released from the planar wafer; and    -   (5) optionally treating the support material to improve its        attachment properties.

Many methods of chemically treating or physically altering the supportmaterial may be used for the optional step (5) to facilitate theattachment of an analyte, such as a test sample and/or a probe used inmultiparameter experimental analysis, to the support 1, 1′. Thetreatment of the supports 1, 1′ can be performed after the release fromthe wafer as described above or alternatively prior to the release fromthe wafers or earlier in the manufacturing process steps. Alternatively,the treatment of the support material layer, step (5), could be omitted.

FIG. 2 provides an illustration of how analytes, namely primary orsecondary analytes 12, 12′, are attached to a section 11 of the support1, 1′. As described in the foregoing, the analytes 12, 12′ may be eitherprobes or target analytes in test samples depending on how experimentsutilizing the supports 1, 1′ are designed and customised. Differenttypes of analytes 12, 12′ may be attached to supports 1, 1′ fabricatedby steps (1) to (5) above, either before or after executingphotolithographic operations or releasing the supports 1, 1′ from theplanar wafer. By modifying the surface 6, 6′ of the supports 1, 1′ orthe analytes 12, 12′, the attachment between analytes 12, 12′ andsupports 1, 1′ is improved. Anodising the attachment surface 6, 6′ ofthe supports 1, 1′ is one way of providing such improved attachmentenhancement. Aluminium is a preferred material for the supports 1, 1′and there are known methods of growing porous surfaces through aluminiumanodisation to improve the attachment properties thereof. Likewise,processes for sealing such porous surfaces are also known. The Applicanthas exploited such knowledge to develop a relatively simple process forgrowing an absorbing surface having accurately controlled porosity anddepth. Such porous surfaces 6, 6′ are capable of achieving a mechanicalbinding to preferred analyte 12, 12′. Using an avidin-biotin system isanother approach for improving chemical binding between the supports 1,1′ and their associated analytes 12, 12′. The supports' 1, 1′ surface 6,6′ may also be treated with a binding material such as silane and/orbiotin, to further enhance attachment properties. The supports 1, 1′preferably have silane baked onto their surfaces 6, 6′. Attachinglinking molecules, for example avidin-biotin sandwich system, to theanalytes 12, 12′ further enhances their chemical molecular attachmentproperties.

The enhanced attachment is preferably achieved through having covalentbonds between attachment surface 6, 6′ of the support 1, 1′ and theanalytes 12, 12′. The covalent bonds prevent the analytes 12, 12′ frombeing dislodged from the supports 1, 1′ and causing disturbingbackground noise during analysis. There is also a potential problem thatloose analytes 12, 12′ could prevent the identification of reactionsthat have occurred. It is found to be important to wash the activesupports 1, 1′, said supports 1, 1′ having analytes 12, 12′ attachedthereto, after attachment to remove any excess analytes 12, 12′ thatcould otherwise increase the noise in the experiment during analysis.Discrimination of the tests is thereby enhanced through a bettersignal-to-noise ratio.

The primary support 1 and/or the secondary support 1′ described aboveutilises the benefits of a cost effective manufacturing technique withthe possibility to tailor the design and identification coding asrequired. These benefits allows for the largest dimension 3 of thesupport 1, 1′ to be circa 500 μm or less, preferably circa 300 μm orless, more preferably circa 150 μm or less, most preferably circa 100 μmor less, circa 50 μm or less, or even circa 10 μm or less in length. Byattaching a different primary analyte 12, 12′ to each support 1, 1′ witha specific identification code 2, 2′, a large number of analytes 12,12′, such as molecules or other appropriate compounds, can be preparedfor testing. As described in the foregoing, the shape as well as thesize of the supports 1, 1′ may be varied as appropriate usingmicrofabrication manufacturing techniques. Non-exhaustive examples ofpossible shapes are, for example, circular, elliptical, elongated,square, rectangular, multi-cornered or even multi-layered supports ofthe same or different materials. It is also, in some applications,preferable to have the primary supports 1 and/or the secondary supports1′ in the size of nanoparticles with a largest dimension of circa 500 nmor less; examples of such nanoparticles comprise cylindrical nanobars.However, a lower limit to size is governed by sufficient sensitivity ofthe reaction kinetics being achieved.

In FIG. 3, a multiparameter analysis assay reaction 13 is depicted. Theassay reaction 13 occurs in fluid solution according to a firstembodiment of the invention. Preferably, the solution is a liquid whichimproves the sensitivity of the assay reaction. The assay 13 includesseveral suspended primary supports 1 covalently bound to primaryanalytes 12, such as target molecules. In the assay 13, there are alsoseveral suspended secondary supports 1′ covalently bound to secondaryanalytes 12′, such as test molecules. Many different target molecules 12and test molecules 12′ are used for functioning as reaction molecules inan associated experiment to be performed, with each type of targetmolecule 12 being attached to a primary support 1 with a specificidentification 2 and each type of test molecule 12′ being attached to asecondary support 1′ with specific identification 2′. If there is amatch between one or more target molecule 12 and one or more testmolecule 12′, they will mutually bind, preferably through a hydrogenbond, to generate a new dual support unit 16. The specificidentification 2, 2′ on both the supports 1, 1′, can then be determinedto indicate which target molecule(s) 12 and test molecule(s) 12′ haveinteracted. This system and methodology allows the analysis of multipleparameters in the same experiment. In the past, experiments have beenlimited to a very small number, often two to four, of fluorescentlabelled target molecules. The simultaneous testing limitation has beendue to the high risk of spectral overlap of the fluorescent labels. Theutilisation of multiple supports 1, 1′ allows much greater numbers ofmultiplexing including multiple target molecules 12 and test molecules12′.

By utilising the secondary supports 1′ instead of traditional reactiontags the benefits of being able to multiplex and customise the secondaryanalytes (target analytes) doubles compared to the traditional multipleparticle based arrays tested against a single target analyte. With theidentification of the secondary analyte 12′ through a secondary support1′ a better reaction signal may be achieved. Also when the number ofmultiplexed analytes increases there are increasing problems with crossreactivity. In running a conventional particle array experiment crossreactivity between e.g. 1000 primary analytes verses 1 secondary analyteis more problematic than running an experiment using e.g. 100 primaryanalytes 12 on identifiable primary supports 1 verses 10 secondaryanalytes 12′ on identifiable secondary supports 1′. This allows verycomplicated biological systems with multiple dimensional features to beanalysed through experiments rather than only in silica modelling.

An example of the methodology of performing these simultaneous testingaccording to this embodiment will now be described. The methodologyinvolves a first step of providing several primary supports 1 withappropriately attached drug targets 12 suspended in a liquid solution.In a second step of the methodology, a suspension of several secondarysupports 1′ with appropriately attached test compounds 12′ are added tothe solution; such addition allows many different test compounds 12′ tobe tested against many different target molecules 12 simultaneously,thereby dramatically improving testing throughput in comparison tocontemporary methods. The resulting support units 16 are then detectedby a measuring apparatus. In FIG. 3, it is also shown that the secondarysupports 1′ are potentially smaller than the primary supports 1. Such adifference in size is capable of improving the reading of the primarysupports' 1 identification 2, because the secondary supports 1′ do notinterfere with, for example, a transmission barcode identification 2.There is also a reduction in the use of reagents as many more compoundsare tested against many molecules simultaneously than what has beenpossible in the past. Preferably, each support 1, 1′ with a uniqueidentification has only one type of analyte 12, 12′, e.g. a specificprotein, attached thereon. It would however be possible to have morethan one type of analyte 12, 12′ attached to each support 1, 1′ with aunique identification if multiple reactions were analysed on a support1, 1′. As the analytes 12, 12′ are preferably attached to the supports1, 1′ in solution the whole of the supports 1, 1′ are covered allowinggood experimental sensitivity.

When performing a multiparameter analysis of analytes experiment, manydifferent types of analytes 12, 12′ may be used. For the life scienceindustry, the analytes 12, 12′ may be antibodies, antigens, proteins,enzyme substrate, carbohydrates, peptides, nucleic acids, peptidenucleic acids, cell lines, chemical components, oligonucleotides, serumcomponents, drugs or any derivatives or fragments thereof. Themultiparameter analysis system is very useful in the area ofprotein-protein interaction as there are very large numbers of proteinswhose interaction needs to be investigated. For other industries, theanalytes can be, for example, dyes, preservatives, labelling chemicals(for example for tracking the movement of counterfeit products),radioactive labelling chemicals, and food.

In FIGS. 4 a and 4 b, there is shown schematically the interaction of amatching pair of primary support 1 with bound primary analyte 12 andsecondary support 1′ with bound secondary analyte 12′ pre- andpost-reaction. These primary and secondary supports 1, 1′ each hasbar-coded identifications 2, 2′ as described above. In this embodiment,the analytes 12, 12′ have been added to the supports prior to theirrelease from their corresponding planar wafer during manufacturing,thereby resulting in the analytes 12, 12′ only being present on one sideof the supports 1, 1′. It is also possible to achieve a similar effectby coating one surface of the supports 1, 1′ with a material thatprevents the analytes 12, 12′ binding thereto. This coating furtherensures that the primary and secondary supports only interact with aspecific main surface 7, 7′ facing each other, thereby enabling therespective identification codes 2, 2′ to be read suitably in reflectionor transmission with satisfactory signal-to-noise ratio. Ifcircumstances arise where it would be desirable to separate the supportunits 16 from non reacted supports 1, 1′, a barrier sorting can beemployed based on size or a sorting arrangement well known in the art ofcell sorting is suitably used.

In FIGS. 5 a and 5 b, there is shown schematically an alternativeembodiment of the multiparameter analysis system using supports 1, 1′with different identification features 2, 2′. In this alternativeembodiment, the primary support 1 is of cuboid shape and includes aradio frequency transponder identification (RWID) 2. The secondarysupport 1′ has a colour coded identification 2′. In FIG. 5 b it can beseen how the primary analyte 12 on the primary support 1 binds directlyto the secondary analyte 12′ on the secondary support 12′. The benefitof using a colour coded support rather than just a colour label as intraditional sandwich assays is that patterns or colour variations can beused to increase the codes possible, such as the Luminex xMap system.When the primary support 1 is made much larger than the secondarysupports 1′, for example by at least a factor of 5, it becomes easier tomeasure quantitatively the amount of secondary analyte 12′ present inthe sample provided that the amount of secondary analyte 12′ persecondary support 1′ is known. An alternative approach is to measureonly yes/no interactions between the different supports 1, 1′. It isalso preferable to use two different types of identification means 2, 2′in the multiparameter analysis as there is less risk of the interferencebetween the different identification signals.

The reader used for reading the supports 1, 1′ that have interacted toform a dual support units 16 is a modified version of the readerdescribed in detail later in the detailed description with reference toFIG. 8. If the dual support units have different types of identificationfeatures 2, 2′ for the primary or secondary supports 1, 1′ a readingunit 30 capable of detecting two different identification signals mustbe used; the reading unit 30 is described later in FIG. 8. If the sametype of identification means 2, 2′, such as barcodes, is used for theprimary and secondary supports 1, 1′, the reader unit 30 needs to beadapted to allow reading of the both identification featuressimultaneously. For the embodiment described in FIGS. 4 a and 4 b,support 1, 1′ interrogation is potentially achievable by including twoor more sub-readers in the reader unit 30 arranged mutually diagonallyopposite to read the barcodes simultaneously in reflection ortransmission. If large numbers of dual support units 16 are to beanalysed, the thermodynamic principles of flow cytometry add thepossibility of very high throughputs of up to several thousand units 16per hour. Once again, the reader unit 30 is preferably then arranged todetect the supports' 1, 1′ identification features 2, 2′. The use oflasers for the reader unit 30 allows very small features of the supportunit to be read, for example features of lateral size comparable to thewavelength of laser radiation employed.

In FIG. 6, there is shown schematically a system according to anadditional embodiment of the multiparameter analysis system. The systemis indicated generally by 17 and comprises a substrate (array) 18accommodating a quantity of liquid solution 19 including supports 1, 1′.The substrate 18, which hereinafter also is referred to as an array ormicroarray, has two substantially planar main surfaces 20 and can be ofany desired shape, but is more preferably rectangular, for examplesubstantially square. The substrate 18 may also be made of a variety ofmaterials, such as glass, metal, plastics materials, wafers, membranesor any other material contemporarily used for fabricating microarrays.Most preferably, the substrate 18 is fabricated from a material, forexample glass (microscope slide) or plastics material (for example anacrylate), which is light transmissive. Such material characteristicspotentially enable a support 1, 1′ with a transmissive bar-codeidentification feature 2, 2′ to be read in transmission whilst on thesubstrate 18. The substrate's 18 top main surface 20 is preferablyplanar or may be divided into sections by partitioning features, forexample wells or boundaries, to prevent cross contamination betweensections. The main surface 20 of the substrate 18 has preferably asurface area in a range of 0.25 cm² to 50 cm², more preferably in arange of circa 1 cm² to 25 cm² and most preferably in a range of circa 2cm² to 6 cm². The liquid 19, which is placed on the substrate 18, isappropriately a liquid buffer solution and is normally an aqueous basedsolution. The system 17 can be considered to be an assay comprising theliquid solution 19 with loaded supports 1, 1′ placed on a substrate 18.The system 17 is of considerable advantage in that it is capable ofproviding the benefits of using two dimensional substrates 18 withestablished reader technology, multiplexing as well as the advantages ofthe multiparameter analysis system using microcarriers with higherthroughput, good sensitivity and satisfactory reaction kinetics.

In a further embodiment of the invention, specific test molecules 12′are attached to individual supports 1′ preferably through covalentbonds. Multiple test molecules 12′ can be tested for their affect on theactivity of multiple target molecules 12 attached to individual supports1 by placing the liquid solution 19 with suspended supports 1, 1′ on asubstrate 18 with molecules 21 that are interrogating the targetmolecules 12 attached thereto. An example of the additional embodimentof the invention is employing a microarray pre-spotted with substratemolecules 21 to simultaneously test the activity of multiple testmolecules 12′ attached to individual supports 1′ against multiple targetmolecules 12 attached to individual supports 1. A bonding reactionoccurs when the molecules 21 do bind to the test molecule 12 and/or thetarget molecule 12′. The results of the reaction between test molecules12′ and target molecules 12 will be based on the final position of thesupports 1, 1′ together with their identification code 2, 2′.

In FIGS. 7 a and 7 b, an assay reaction indicated generally by 14 isdepicted which takes place on a substrate 18 according to the previousembodiment of the invention. The assay 14 consists of a liquid solutionwith suspended supports 1, 1′ and analytes 12, 12′. The analytes 12, 12′are made up of target molecules 12 and test molecules 12′. Manydifferent target molecules 12 and test molecules 12′ are used forfunctioning as reaction molecules in the experiment to be performed,with each type of target molecules 12 being attached to a primarysupport 1 with a specific identification 2, and each type of testmolecules 12′ being attached to a secondary support 1′ with a specificidentification 2′. The supports 1, 1′ preferably with at least onecovalently bound target molecule 12 or test molecules 12′, thereon, aresuspended in the liquid solution 19, which is then is placed on a mainsurface 20 of the substrate 18. The substrate 18 further has tertiarymolecules 21, which act as substrates for the target molecules 12, 12′,bound to the main surface 20 through, e.g. a covalent bond. Thispotentially allows the use of a pre-spotted microarray as the substrate18 in the system 17 to add another dimension to the multiparameteranalysis. The molecules 21 bound to the substrate are suitably labelledwith a fluorophore 22 and quencher molecule 23.

If there is a match between one or more target molecule(s) 12 and testmolecule(s) 12′, they will mutually bind, preferably through a hydrogenbond, to generate a new dual support unit 16. If the test molecule 12′inhibits the interaction of the target molecule 12 with its substratetertiary molecule 21, the quencher molecule 23 will not be cleaved fromthe substrate molecule 21, thus the fluorophore 22 will remain quenchedas shown in FIG. 7 a. If, however, the test molecule 12′ binds to thetarget molecule 12, but does not inhibit the interaction of the targetmolecule with its substrate molecule 21, the quencher molecule will becleaved and the fluorophore will emit a fluorescent signal whenoptically interrogated.

An example of this embodiment is the use of several supports 1 withappropriately attached enzyme targets suspended in a liquid solution.When performing an assay, a suspension of several supports withappropriately attached test compounds are added to the solution.Molecules that are known to be substrates for the enzymes would bepre-spotted onto the array substrate 18 at predefined positions. Thisallows many different test compounds to be tested against many differentenzyme target molecules simultaneously to indicate not only whether ornot the test compounds bind the target enzymes, but also the effect ofsaid binding on the activity of the target enzymes.

Appropriate identification of supports 1, 1′, as mentioned above, refersto the importance of using a specific identification for a specificanalyte 12, 12′, for example the target molecule 12 or the test molecule12′. Such an arrangement also allows the use of predeterminedidentification codes 2, 2′ for certain analytes 12, 12′ but will alsoallow for matching of identification codes 2, 2′ and analytes 12, 12′ asdesired when designing an experiment.

When performing tests of multiple target molecules 12 against multipletest molecules 12′, as in the described embodiments of the invention, itis also of benefit to analyse the experiments at different time points.This temporal analysis is potentially useful in pharmaceutical profilingwhere changes over time are important to record.

A reading system used for reading the substrate 18 with loaded supports1, 1′ suspended thereon in a liquid solution 19 will now be describedwith reference to FIGS. 6, 7 a and 7 b.

Laser, ultra violet (UV) or light emitting diode (LED) reader equipmentcurrently used for the analysis of, for example, microarrays ormicrocarrier-based assays is also susceptible to being employed with theaforementioned system for analysing multiple parameters of analytes 12,12′. In the system, test results of reacting analytes 12, 12′ aremeasured as a yes/no binary result or by the degree of fluorescenceemitted from a signal emitting label 23.

The system is indicated generally by 24 in FIG. 8 and comprises areader. The reader includes a measuring unit indicated by 25 formeasuring activity of the supports 1, 1′ tagged to analytes 12, 12′. Themeasuring unit-25 has a detection unit 27 to detect the fluorescentreaction signal from unquenched substrates 22 and a reader unit 30 toread the identification code 2, 2′ of the supports 1, 1′. The detectionunit 27 has a fluorescence microscope for detecting the fluorescentsignal indicating reaction. The reader unit 30 has a barcode reader toread the transmissive bar-codes 2, 2′ of the supports 1, 1′. It ispreferable to have different type of signal for the support 1, 1′identification 2, 2′ and the reaction detection, as there then is alimited risk of the signals being mixed up or being overlapping(spectral overlap). This allows for greater multiplexing (multiplesimultaneous reactions) possibilities.

Once a sufficient number of supports 1, 1′ have been read, a processingunit 28 of the measuring unit 25 calculates the results of the testsassociated with the supports 1, 1′. This sufficient number is preferablybetween 10 and 100 copies of each type of supports 1, 1′; this number ispreferably to enable statistical analysis to be performed on testresults. For example, statistical analysis such as mean calculation andstandard deviation calculation can be executed for fluorescenceassociated with the unquenched fluorophores 22. A processing unit 28 isalso included for controlling the detector and reader units 27, 30 sothat the each individual support 1, 1′ is only analysed once.

Normally, all the supports 1, 1′ on the substrate 18 are analysed toverify the total quality of the experiment. In cases where there couldbe an interest in saving time and/or processing capacity, the softwareof the processing unit 28 can preferably be configured to analyse onlythe supports that have interacted and emit a signal, indicating that aninteraction between characteristics of the analytes 12, 12′ hasoccurred. The analysis of the loaded substrate 18 using the measuringunit 25 is a very cost effective, easy to perform and suitable way tomultiply the analysing capacity for low to medium sample numbers in therange of, for example, single figures to a few thousand supports 1, 1′on each substrate 18.

Preferred paths 50 for systematically interrogating the substrate 18 areshown in FIGS. 9 a and 9 b. FIG. 9 a is a depiction of a meander-typeinterrogation regime, whereas FIG. 9 b is a depiction of a spiral-typeinterrogation regime. There are of course many other possible paths 50apparent to one skilled in the art, for example moving the substrate 18in an opposite direction to the path 50, moving the substrate in ameandering diagonal path, or covering the whole substrate in onesubstantially linear path across its surface. However, the regimes ofFIGS. 9 a, 9 b are efficient for achieving an enhanced support 1, 1′read speed. A stepper-motor actuated base plate 40 supporting andbearing the substrate 18 may be used to move the substrate 18 aroundwhile the measuring unit 25 is held stationary. The most preferablemethod of analysis would, however, be to move the measuring unit 25while the substrate is held stationary. The positions of supports 1, 1′are tracked so that they are analysed once only.

The measuring unit's 25 reader unit 30 for image-processing is used tocapture digital images of each field of the substrate 18 with a liquidsolution 19 suspending supports 1, 1′ with attached analytes 12, 12′thereon. Digital images thereby obtained correspond to light transmittedthrough the substrate 18 and past a base plate 40 and then through thesupports 1, 1′ rendering the supports 1, 1′ in silhouette view; suchsilhouette images of the supports 1, 1′ are analysed by the reader unit30 in combination with a processing unit 28. The sequentialidentification 2, 2′, for example a bar-code, associated with eachsupport 1, 1′ is hence identified from its transmitted light profile bythe reader unit 30. The signal emitting unit 29 generates a fluorescentsignal, which signal makes the fluorophores 22 on the substratemolecules 21 fluoresce indicating a positive reaction.

The processing unit 28 is connected to the light source 45, the signalunit 29, the reader unit 30, and the detector unit 27 and to a display46. Moreover, the processing unit 28 comprises a control system forcontrolling the light source 45 and the signal unit 29. The lightsilhouette and fluorescent signals from the fluorophores 22 on thesubstrate molecules 21 pass via an optical assembly 41, for example anassembly comprising one or more lenses and/or one or more mirrors,towards the detector unit 27 and reader unit 30. A mirror 42 is used todivide the optical signals into two paths and optical filters 43, 44 areused to filter out unwanted optical signals based on their wavelength.Alternatively, the light source 45 and signal unit 29 can be turned onand off at intervals, for example mutually alternately. Signals arereceived from the reader unit 30 and detector unit 27, which areprocessed and corresponding statistical analysis results presented on adisplay 46. Similar numbers of each type of supports 1, 1′ are requiredto give optimal statistical analysis of experiments. Such statisticalanalysis is well known in the art.

The intended uses of the system 17 may be in any process whereexperiments requiring the analysis of three dimensional multiparameteranalysis of analytes. The applications where several parameters areinvolved are for example in biochemical detection of one or more analytecharacteristics including, lead target identification and drugtargeting. There will be many other applications for this system foralternative industries requiring multiparameter analysis of analytes.

Flow based reading of the experiments using primary and secondarysupports 1, 1′ as identification of binding characteristics of theprimary and secondary analytes 12,12′ provides an alternative orcomplement to the planar reader described previously and a fast andefficient way of analysing the reaction results. Sorting and detectionof experiments using the primary and secondary supports 1, 1′ have beenperformed at a rate of ca 20 supports/second on the Union Biometrica,COPAS™, flow cytometer. The forward scatter and measurements of e.g.length and density give good indication of any interaction between theanalytes 12, 12′ on the primary and the secondary supports 1, 1′. Sheathfluid in the flow cytometer focuses the supports 1, 1′ to the centre ofa flow channel and allows detection using two lasers, which to cover thecross section the flow channel and arranged at ca 90 degrees to eachother and with a joint focal pint at the centre of the channel. Bothqualitative and quantitative results may be measured. Other flow readerswhich work well for analysing the multiparameter experiments using theembodiments of the primary and secondary supports 1, 1′ described arefrom DakoCytomation (MoFlo™) and Becton Dickenson (FACScan™).

It will be appreciated that modifications can be made to embodiments ofthe invention described in the foregoing without departing from thescope of the invention as defined by the appended claims. For example,when a conventional spotted microarray 18 or ELISA well plate withsubstrate molecules 21 attached directly to the array's 18 surface 20 isused as the array (substrate) 18 with positional identification in thesystem 14, the fluorophore 22 and quencher molecule 23 can be arrangedto deactivate the fluorescent signal when a dual support unit 16 reactswith a suitable tertiary molecule 21.

1. A system for multiparameter analysis of analytes, the systemcomprising: (a) primary supports with a largest dimension of 500 μm orless suspended in use in a fluid solution, wherein each primary supportcomprises identification means for identification thereof, and at leastone primary analyte is bound to each primary support; (b) a secondaryanalyte suspended in use in the fluid solution; and (c) measuring meansarranged in communication with the fluid solution for monitoringinteraction between the primary analyte and secondary analyte,characterised in that: (d) secondary supports with a largest dimensionless than or equal to the largest dimension of the primary supports aresuspended in use in the fluid solution, wherein each secondary supportcomprises identification means for identification thereof, and at leastone secondary analyte is bound to each of the secondary supports; and(e) the measuring means is arranged to detect any post-reactioninteraction between one or more primary analytes and one or moresecondary analytes by detecting the identification means of the primaryand secondary supports attached thereto.
 2. A system according to claim1, wherein the largest dimension of the primary support is less than 300μm.
 3. A system according to claim 2, wherein the largest dimension ofthe primary support is less than 150 μm.
 4. A system according to claim3, wherein the largest dimension of the primary support is less than 50μm.
 5. A system according to claim 1, wherein the largest dimension ofthe secondary support is less than that of the primary support.
 6. Asystem according to claim 5, wherein the largest dimension of thesecondary support is less than 100 μm.
 7. A system according to claim 6,wherein the largest dimension of the secondary support is less than 50μm.
 8. A system according to claim 7, wherein the largest dimension ofthe secondary support is less than 10 μm.
 9. A system according to claim1, wherein at least one of the identification means comprises one ormore distinguishing geometrical features, such as shape, size, barcodeor dotcode, enabling identification of each support.
 10. A systemaccording to claim 1, wherein at least one of the identification meansis a radio frequency identification transponder (RFID).
 11. A systemaccording to claim 1, wherein at least one of the identification meansis an optical identification, such as fluorescence or colour based. 12.A system according to claim 1, wherein the primary or secondary supportsare only partially covered in their respective primary or secondaryanalyte.
 13. A system according to claim 1, wherein the fluid solutionis a liquid.
 14. A system according to claim 13, wherein the liquidsuspension is accommodated on a solid substrate, which substrateincludes a main surface extending substantially in a two dimensionalplane and has tertiary analytes fixedly arranged thereon for positionalidentification.
 15. A method of multiparameter analysis of analytes, themethod including the steps of: (a) providing at least one primarysupport with a largest dimension of 500 μm or less and withidentification means for identification thereof; (b) binding at leastone primary analyte to each primary support; (c) suspending the primarysupport with its primary analyte and a secondary analyte in a fluidsolution; and (d) providing measuring means in communication with thefluid solution for monitoring interaction between the primary analyteand the secondary analyte, characterised in that the method furthercomprises the steps of: (e) providing secondary supports with a largestdimension less than or equal to the largest dimension of the primarysupports and with identification means for identification thereof; (f)binding at least one secondary analyte to each of the secondarysupports, (g) suspending the secondary supports in use in the fluidsolution, and (h) arranging for the measuring means to detect anypost-reaction interaction between one or more primary analytes and oneor more secondary analytes by detecting the identification means of theprimary and secondary supports attached thereto.
 16. (canceled) 17.(canceled)